Assay procedure using fluorogenic tracers

ABSTRACT

Fluorescent energy transfer dyes capable of moving between a more stacked configuration to exhibit fluorescent quenching and a more spaced configuration to exhibit fluorescence can be conjugated to a peptide epitope or nucleic acid for use in the detection of an unknown antibody in bulk solution. The resulting labeled peptide reagent can be used in an immunoassay procedure by placing it in bulk solution along with the unknown antibody to be detected. When the antibody binds to the peptide epitope, the pair of dyes carried by the peptide epitope will have their configuration altered from a stacked to an unstacked configuration and will exhibit a fluorescent increase in response thereto.

RELATED APPLICATION

This application is a continuation of application Ser. No. 08/484,563,filed Jun. 7, 1995 now abandoned and a CIP of Ser. No. 08/096,338, filedJul. 23, 1993.

TECHNICAL FIELD

This invention relates generally to biological assays, and morespecifically to assay reagents labeled with fluorescent materials whichreagents can be “toggled” from an intramolecular dimer to a fluorescentmonomer by antibody binding.

STATE OF THE ART

Most clinical assays (e.g., immunoassays, DNA probe assays) areheterogeneous and consist of at least two steps: the binding of anantigen to its antibody, followed by physical separation of the boundfrom free antigens. In some more sensitive assays (e.g., “ELISA” or“EIA”) multiple steps are required. Homogeneous immunoassays, on theother hand, can distinguish between bound antigens and free ones withoutthe need of additional separation steps. They are simple, rapid, yetmore precise, more cost effective, and have the potential for totalautomation. For these reasons, separation-free assays are preferred inmany applications such as biosensors, bioprobes and other automatedinstrumentation. J. P. Gosling, Clin. Chem., 36:1408-1427 (1990), D. W.Chan and M. T. Perlstein, Eds., Immunoassay, A Practical Guide (AcademicPress, New York, 1987), and E. F. Ullman and P. L. Khanna, Methods inEnzymology, 74:28-60 (1981).

However, because of various technical complications homogeneous systemshave been difficult to obtain, with the exception of a few assayssuitable only for small molecules. J. F. Burd et al., Clin. Chem.,23:1402-1408 (1977), M. E. Jolley et al., Clin. Chem., 27:1190-1197(1981), and D. L. Morris et al., Anal. Chem., 53:658-665 (1981).

It would be an improvement in the art to develop and characterize newfluorogenic tracer antigens that can be used as “reporter molecules” forthe binding event without the need of separation steps and the labelingof antibodies. The development of such tracers could greatly facilitatethe automation of a large array of clinical assays, especially of highmolecular weight antigens. It would help reduce the operational time andcost, and make such assays more readily accessible to doctors andpatients. Also, such tracers would be extremely useful for rapidlyscreening large numbers of recombinant antibodies generated with geneticengineering techniques, such as those described in C. F. Barbas et al.,Proc. Natl. Acad. Sci. USA 89:4457-4461 (1992), R. A. Lerner et al.,Science 258:1313-1314 (1992), and Marks et al. J. Biol. Chem.267:16007-16010 (1992).

DISCLOSURE OF THE INVENTION

The invention includes a fluorogenic tracer antigen that obviates theneed for separation steps or the labeling of antibodies in theperformance of an assay. The tracer is a short antigen-mimicking peptidelabeled with both a fluorescent energy transfer donor and fluorescentenergy transfer acceptor molecules. When free in solution, the tracerexhibits very low fluorescence due to intramolecular dye dimerization.After binding to an antibody of the native antigen, fluorescence issignificantly enhanced as a result of the dissociation of intramoleculardimers brought about by conformational changes in the tracer peptide.

The invention thus includes an immunoassay procedure for detecting andquantifying unknown analyte antibody or analyte antigen in bulksolution, a reagent for use in such an immunoassay procedure, and aprocess for making such a reagent. The reagent which is used in thepresent procedure is a peptide epitope that is recognized by theantibody in bulk solution, the peptide epitope conjugated to a pair offluorescent energy transfer dyes capable of moving between a stackedconfiguration to exhibit fluorescence quenching and a spaced (unstacked)configuration to exhibit fluorescence.

A procedure for using the previously described reagent in a homogeneousantibody assay includes: placing in bulk solution a conjugate of apeptide epitope for the unknown amount of analyte antibody, and a pairof fluorescent dyes. This reagent is capable of moving between a stackedconfiguration to exhibit fluorescent quenching and a more spacedconfiguration to exhibit fluorescence. Also placed into the bulksolution is the unknown antibody which will bind with the peptideepitope which is conjugated to the pair of dyes. When this binding eventoccurs, the configuration of the pair of dyes will be altered between aninitial stacked configuration (when in solution) to an unstackedconfiguration, when the epitope is bonded to the antibody, with aconcomitant increase in fluorescent energy in response to the binding.

It is also within the contemplation of the invention to design ahomogenous antigen assay or a homogenous DNA (or RNA) probe assay.

The tracers have uses including homogenous detection of macromolecules(e.g. antibodies, antigens, DNA, and RNA) of clinical interest and rapidscreening of recombinant antibodies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a doubly labeled oligopeptide useful in the practice ofone embodiment of the invention.

FIG. 2 is a graph depicting the absorption spectra of the FpepTconjugate (solid line).

FIG. 3 is a graph depicting the relative fluorescence intensity vs.wavelength.

FIG. 4 is a graph depicting the relative fluorescence intensity vs.wavelength.

FIG. 5 graphically depicts the technical fluorescence spectra of FpepT(10⁻⁷ M) as 5 μl aliquots of stock anti-hCG Mab were added. The totalanti-hCG concentration changed from zero to 2.6×10⁻⁷ M at an incrementof 3.7×10⁻⁸ M with a dilution factor of 2%.

FIG. 6 is a comparison of the absorption spectra of FpepT (6.5×10⁻⁶ M)in the absence (dashed) and presence (solid) of anti-hCG antibodies(7.5×10⁻⁶ M).

FIG. 7 depicts graphically the fluorescence enhancement factor E as afunction of antibody concentration (EXX=493 mn and EM=590 nm).

FIG. 8 graphically depicts the fluorescence intensities as a function ofFpepT concentration (EX=561 nm and EM=590 nm).

FIG. 9 graphically depicts the exchange of FpepT by hCG (EX=561 nm andEM=590 nm) in an antigen assay according to the invention.

FIG. 10 is a graph depicting the fluorescence anisotropy of FpepT(1.6×10⁻⁸ M).

FIG. 11 depicts the chemical structure of a dimeric conjugate consistingof fluorescein and tetramethylrhodamine (“TMR”) linked via a hexanespacer in a model system.

FIG. 12 is the absorption spectra of the conjugate shown in FIG. 11.

FIG. 13 depicts the technical fluorescence spectra of the FhexTconjugate (4.35×10⁻⁷ M) as aliquots of (Fab′)2 fragment ofantifluorescein 4-4-20 were added.

FIG. 14 schematically represents an embodiment of the invention.

FIG. 15 is a schematic illustration of an embodiment of the invention ofusing antifluorescein antibodies (anti-F) to modulate rhodaminefluorescence. Fluorescein (F) and tetramethylrhodamine (T) arechemically linked via a short alkyl spacer. Binding of anti-F to thisbichromophoric conjugate drives the intramolecular dimer-monomerequilibrium towards the monomer side, which results in a concomitantincrease in rhodamine fluorescence. This process is specific and can bereversed by the addition of fluorescein and its analogs such as5-aminofluorescein (AF).

FIG. 16 depicts chemical structures of six bichromophoric conjugatesconsisting of fluorescein and tetramethylrhodamine linked by varyingnumber of CH₂ units using different chemistry or isomers.

FIG. 17 depicts absorption spectrum of F—(CH₂)₆—T (III) (solid line).The dashed line shows a multiple linear regression fit of the measuredspectrum to a model assuming simple summation of single-labeledconjugates. Discrepancies between these two curves (poor fit) indicatesignificant ground-state interactions exist between F and T in theconjugate. Similar results were obtained for conjugates I, II, IV, VI(data not shown).

FIG. 18 is a graph depicting absorption spectrum of F—(CH₂)₆—T (V) whichuses the 6-isomers of F and T, demonstrating intramoleculardimerization. The bands at 515 nm and 555 nm are characteristic of thespectral splitting due to ring stacking as often observed for rhodaminehomo-dimers. In this particular case, however, the ring stacking isbelieved to be due to the formation of fluorescein-rhodaminehetero-dimers, instead of homo-dimers.

FIG. 19 is a graph depicting technical fluorescence spectra ofF—(CH₂)₆—T (V) (4.35×10⁻⁷ M) as aliquots of (Fab′)₂ fragment ofantifluorescein 4-4-20 were added. Ex=475 nm. The total (Fab′)₂concentration varied from zero to 2.6×10⁻⁷ M. Measurements were made in100 mM phosphate buffer, pH 7.4 at 25° C. After each addition of (Fab′)₂aliquots, samples were stirred for 2 min before measurements were takento allow for equilibration. The overall titrating volume was less than4% of the total sample volume.

FIG. 20 is a graph depicting fluorescence enhancement factor (E)calculated from intensities at 576 nm (λ_(max)) in FIG. 19 as a functionof (Fab′)₂ concentration (P_(o)) A value of 6×10⁻¹⁰ M was obtained forK_(d) and 28.5.

FIG. 21 is a graph depicting technical fluorescence spectra ofF—(CH₂)₆—T (V) (4.2×10⁻⁷ M) in the presence of 4-4-20 (Fab′)₂ (2.6×10⁻⁷M) as 10 ml aliquots of 5-aminofluorescein (AF) solution were added. Thetotal AF concentration varied from zero to 6.2×10⁻⁷ M. Spectra measuredafter 2 and 30 min were completely superimposable, indicating thatexchange equilibrium was achieved in a period of less than 2 min.Experimental conditions are the same as in FIG. 18.

FIG. 22 is a graph depicting fluorescence enhancement factor (E) at 589nm as a function of 4-4-20 (intact IgG) concentration (P_(o)) forconjugates I (n=2, filled squares), II (n=4, open squares), III (n=6,open circles) and IV (n=8, filled circles). The maximal enhancementfactor (E^(m) ⁵⁸⁹) was found to be 114.6, 108.4, 41.8 and 26.2,respectively. The data were derived from titrating a solution of eachF—(CH₂)_(n)—T conjugate (˜10⁻⁸ M) with 5 ml aliquots of a stock 4-4-20solution. Fluorescence intensities were measured at 589 nm with Ex=488nm. Other experimental conditions are the same as in FIG. 18.

FIG. 23 is a graph depicting fluorescence enhancement factor (E) at 589nm as a function of concentration (L_(o)) of conjugates I (n=2, filledsquares), II (n=4, open squares), III (n=6, open circles) and IV (n=8,filled circles).

FIG. 24 is a double-axis plot of K_(a) and E_(m) as a function of numberof CH₂ units in linker according to results obtained from FIG. 20.

FIG. 25 is a graph depicting technical fluorescence spectra ofF—(CH₂)₆—T (VI) (3×10⁻⁷ M) as aliquots of 4-4-20 solution were added.Ex=480 nm. The total 4-4-20 concentration varied from zero to 3×10⁻⁷ M.In contrast to F—(CH₂)₆—T (III & V), the free F—(CH₂)₆—T (VI) conjugatehas significant fluorescence from fluorescein and a shoulder peak due torhodamine. Binding to 4-4-20, however, provides a flip-flop in therelative intensities of F and T.

FIG. 26 is a graph depicting fluorescence enhancement factor (E) at 589nm as a function of 4-4-20 concentration (P_(o)). A solution ofF—(CH₂)₆—T (VI) (1.8×10⁻⁷ M) was titrated with aliquots of stock 4-4-20(filled circles, inset) and IgG+BSA (open circles, inset). Fluorescenceintensity was measured at 589 nm with Ex=488 nm. A value of 5×10⁻⁷ M wasobtained for K_(d).

FIG. 27 is a graph depicting fluorescence polarizadon spectra ofF—(CH₂)₆—T (VI) in the absence (c) and presence (b) of 4-4-20 arecompared to that of rhodamine bound to an antirhodamine antibody (a).

BEST MODE OF THE INVENTION

The “peptide epitope” used herein and to which the fluorescent dyes(preferably energy transfer dyes) are joined, either directly or througha spacer structure, is a relatively small, flexible peptide comprisingalpha-amino acids which are joined together through peptide bonds. Ingeneral, there will be sufficient amino acids (e.g. from about 6 toabout 13 amino acids) in the peptide to allow the peptide to fold uponitself. The term “epitope” is to be understood as relating to thespecific surface of the native antigen (or “antigen”) which isdelineated by the area of interaction with an antibody of interest.

The peptide portion of the peptide epitope is an antigen or anantigen-mimicking peptide. Such a peptide may either be a sequentialepitope which is a continuous sequence of the primary structure of theantigen; or an assembled epitope which consists of amino acids distantin the linear sequence, but brought together by tertiary structurefolding. Several methods have recently emerged that enable rapididentification of high affinity binders for almost any monoclonalantibody. See, e.g. H. M. Geysen et al., Molecular Immunology 23:709-715(1986), R. A. Houghten, et al., Nature 354, 84-86 (1991), K. S. Lam, etal., Nature 354:82-83 (1991) and J. K. Scott and G. P. Smith, Science249:386-390 (1990).

The epitope may be chosen from any of various proteins where determiningthe presence of antibodies to the protein may be useful. These includeepitopes from proteins associated with infectious diseases such ashepatitis B, hepatitis C, herpes simplex, and HIV. Epitopes from otheruseful proteins (such as rhesus factor) may also be used.

While not being bound by one theory of why the invention works so well,it is believed that antigen-mimicking peptides are more viable choicesfor the fluorogenic reporter molecules than their native antigensbecause of their small size. If a protein antigen isfluorescently-labeled, the changes in fluorescence signal (intensity,polarization, etc.) upon binding are relatively small. For this reason,previous homogeneous assays for high molecular weight antigens exhibitedpoor sensitivity. See, e.g. K. Nithipatikom and L. B. McGown, Anal.Chem., 59:423-427 (1987). If fluorescently-labeled oligopeptides areused as tracers, however, significant changes in fluorescence signalupon binding occur presumably due to the oligopeptide's small size andchain flexibility.

The intended structure for the peptide epitope, in a preferredembodiment, can be determined by use of the epitope/mimotope screeningtechniques described in U.S. Pat. No. 4,833,092 to H. M. Geysen. In sucha technique, a plurality of peptides having a defined linear sequenceare synthesized, contacted with the antibody of interest, and thepresence or absence of binding between peptide and antibody isdetermined. The presence of the highest level of binding identifies thepreferred candidate or candidates for the peptide epitope for use inaccordance with the present invention. Once the linear sequence of aminoacids in the peptide epitope has been determined, it is well within theskill of persons in the art to synthesize such a peptide epitope usingsolid state peptide synthesis procedures. References which discuss thevarious synthesis methods available include: Merrifield, J. Am. Chem.Soc., 85:2149-2154 (1963); M. Bodanszky et al., Peptide Synthesis, JohnWiley & Sons, 2d Ed., (1976), and J. Stuart et al., Solid Phase PeptideSynthesis, (Pierce Chemical Company, Rockford, Ill., 3d Ed.), H. Neurathet al., Eds., pp. 104-237 (Academic Press, New York, N.Y. (1976)).Appropriate protective groups for use in such synthesis procedures arealso known. See the above references as well as J. F. W. McOmie,Protective Groups in Organic Chemistry, (Plenum Press, New York, N.Y.(1973)).

For protein antigens of unknown primary sequence or other non-proteinantigens, it is still possible to screen for high affinity binders to anantibody using Geysen's method or other more recent approaches basedupon peptide libraries. See, e.g. the work of R. A. Houghten, et al.,Nature, 354: 84-86 (1991) and K. S. Lam, et al., Nature, 354:82-83(1991).

Once obtained, the selected peptide epitope is then labeled with a pairof fluorescent energy transfer (namely, donor and acceptor) dyes which,when appropriately bonded to the peptide epitope to form a “conjugate”,has the characteristic of dimerizing or “stacking” so as to quench anyfluorescence of both fluorophores. The dye pairs do not necessarily haveto be fluorescence energy transfer donor and acceptors. The type of dyeswhich do exhibit such stacking characteristics when bonded to thepeptide epitope within a sufficiently close proximity to one anotherinclude those dyes which have a generally planar aromatic structure soas to be capable of forming homo- or heterodimers when in solution atconcentrations which are sufficiently high for example, 10⁻³ to 10⁻⁴M).

It is well known that some fluorescent dyes (fluoresceins, rhodamines,cyanines, etc.) form dimers in aqueous solution when they are withinclose proximity of each other.

K. K. Rohatgi and G. S. Singhal, J. Phys. Chem., 70:1695-1701 (1966); K.K. Rohatgi and A. K. Mukhopadhyay, Chemical Physics Letters, 12:259-260(1971); and W. West and S. Pearce, J. Phys. Chem., 69:1894-1903 (1965).Due to the interaction between transition dipoles of the resonatingdimeric structure, these dimers exhibit very low fluorescence quantumyields. I. L. Arbeloa, J. Chem. Soc. Faraday Trans., 2:1735-1742 (1981);I. L. Arbeloa, J. Chem. Soc. Faraday Trans., 2 77:1725-1733 (1981); andI. L. Arbeloa and P. R. Ojeda, Chemical Physics Letters, 87:556-560(1982). The monomers of these dyes, however, are highly fluorescent inaqueous solutions. For this reason, dye dimerization has largely beenregarded as an adverse effect in biological applications. Bailey et al.,J. Pharm. & Biomed. Anal., 5:649-658 (1987). This invention uses thisphenomenon to advantage. If two fluorescent dyes are conjugated to bothends of an antigen-mimicking peptide, it is probable that intramoleculardimers will form because of the planar structure of dyes and the shorteffective distance. This will result in significant fluorescencequenching. Upon binding to its antibody, however, the dye-peptideconjugate is expected to undergo conformational changes to accommodateto the active site. The fluorescence intensity will be enhanced as aresult of dissociation of the intramolecular dimers.

Fluorescent energy transfer dyes of the fluorescein family, such asfluorescein, TMR, rhodamine B, and Texas Red are representative dyes ofthis type. Due to the interactions between the transition dipoles of theresonating dimeric structure, the fluorescent quantum yield of the dimerwill be quite low when no antibody which can bind to the peptide epitopeis present as compared to the significantly higher fluorescence quantumyield in aqueous solution when undimerized after the peptide epitope hasbecome bound to the antibody. In this manner, a homogeneous antibodyassay can be designed wherein labeled peptide epitope is placed insolution and the antibody analyte is added, so that the antibody andpeptide bind, causing the dimerization to decrease with an attendantincrease in fluorescence.

Fluorescein and TMR were used herein as the labels since, among otherthings, they are a well-characterized fluorescence energy transfer pairas well. Because this property may increase the Stoke's shift of thefluorescence emission, it is useful in reducing interference fromscattering or serum fluorescence.

The invention is not limited to using fluorescent dyes. Organic ligandsof some lanthanide metals, such as europium (Eu3+) and terbium (Tb3+)may also be used as labels. E. P. Diamandis, Clin. Chem., 37:1486-1491(1991). In the absence of antibodies, the peptide forms anintramolecular coordination complex with these ions. Such a complex ishighly fluorescent and has fluorescence lifetimes ranging from μs to ms.If the peptide changes its conformation from a folded to a more extendedstate upon binding to its antibody, the coordination bonds with themetal could be broken, thus making the peptide non-fluorescent.Therefore, the fraction of bound and free tracers can be related to thenet change in fluorescence intensities. Molecular dynamics and MonteCarlo simulations of the free peptide have shown that the distancebetween —SH and —NH2 groups is about 10 Å for the energy minimizedconformational states. If coordination ligands, e.g., acetyl acetone ordipyridylamine, are introduced at these positions, they should fallwithin the coordination distance with Eu3+ or Tb3+. The extended lengthof a 13-mer peptide is about 50 Å which is far enough to break thecoordination bond to release the ion, resulting in a dark species.

Solutions for use with the invention are ones in which the labeledpeptide epitope and antibodies can be incorporated. They are generallybuffered aqueous solutions and include buffered normal saline with a pHof 6 to 8.

A homogeneous antigen assay can also be designed wherein the aqueoussolution contains antibodies and doubly labeled peptide epitope boundtogether so that the amount of intramolecular dimer formation is lowthereby producing a high fluorescence signal. The addition of unlabeledanalyte ligand, which binds to the antibody bonded to the peptideepitope, will result in a certain fraction of the doubly labeled peptideepitope being displaced from the antibody bringing about a concomitantdecrease in fluorescence resulting from the formation of dimers in thelabeled peptide epitope as it is displaced from the antibody.

Homogeneous assays can, therefore, be achieved by toggling theintramolecular monomer dimer equilibrium through the antibody-antigenbinding event. For antibody assays, the sample is added to a solution ofdoubly-labeled peptide, and the net fluorescence increase is related tothe antibody concentration.

For antigen assays, the sample is added to a solution of antibody mixedwith the doubly-labeled tracer, and the net fluorescence decrease isrelated to the analyte concentration.

In analogy to the antibody-antigen system, this principle may also beutilized in DNA assays. If a DNA probe is used to link the twofluorophores, hybridizing with its target DNA will bring about atransition from intramolecular dimers to monomers. The target DNA cantherefore be measured from the net increase in fluorescence intensity.

A homogenous DNA or RNA hybridization assay in which a pair offluorophores (e.g. fluorescein and rhodamine, or Cy-3 and Cy-5) isattached to the 5′ and 3′ ends, respectively of an oligonucleotide(between 10 and about 30 nucleotides in length) which is complementaryto a target DNA or RNA sequence that is part of a much larger piece ofDNA (e.g. plasmid or chromosomal DNA) or RNA. The flourescently-labeledoligonucleotide would be mixed with the target DNA or RNA and themixture heated to a temperature high enough to denature the double helix(alternatively, the target DNA could be denatured first, at which pointthe nucleotide could be added). As the mixture cools, theoligonucleotide would hybridize with its target sequence. The unboundform of the fluorescently labeled oligonucleotide would benon-fluorescent because of dimer formation between the two dyes. Uponhybridization however, this dimer would dissociate, resulting in anincrease in fluorescence. As such, this is a homogenous, solution phaseassay because no wash steps are required. Its sensitivity would belimited, however, by the sensitivity of the fluorometer for bulkfluorescence (e.g. one picomolar would be a practical limit with currentinstrumentation).

Greater sensitivity can be achieved by the use of a solid-phaseevanescent assay. In this case, the fluorescently labeledoligonucleotide is attached to a waveguide via a non-stick layer (e.g.polyethylene glycol “PEG”), and the target DNA (or RNA) molecule isintroduced to a sensor after being denatured (e.g. by heat). The assaywould work in the same manner as the previously described homogenous DNAhybridization assay, but would have the advantage of the enhancedsensitivity associated with evanescent fluorescence. Potential detectionlimits would be 0.1 pM for a plastic waveguide sensor, and less than 1femtomolar (fM) for IOW sensors.

Alternatively (to 10 to 30 base pair long DNA or RNA), longer nucleotideprobes could be constructed by attaching the pair of fluorescent dyes tospecially modified nucleotides in the middle of the probe, rather thanat the 5′- and 3′-ends.

In the previously described DNA or RNA assays, nucleotide analogues thatwill hybridize with DNA and/or RNA, but are not degraded by plasmaproteins may be advantageously substituted.

It is broadly within the scope of the present invention to immobilizethe peptide epitope containing the pair of fluorescent energy transferdyes which are capable of moving between stacked and unstackedconfigurations, on a waveguide through which light can be shown togenerate an evanescent wave in the bulk solution. It is within this areaof the evanescent wave that the fluorescent energy transfercharacteristics of the selected dyes can be monitored to appropriatelydetect antigen-antibody binding events and thereby perform theimmunoassay. However, a heterogeneous assay format is not the preferredembodiment. A homogeneous assay is preferred in view of simplicity andthe absence of potential background signals due to the presence of thesupport as is well known to persons of ordinary skill in the art.

The fluorogenic tracer antigen described has many potentialapplications. First, its simplicity, speed, and sensitivity allowadaptation to existing automated instrumentation, such as the 96-wellfluorescence plate reader or the TDX fluorescence polarization analyzer.Second, because labels all reside on the tracer peptide, fluorescentlabeling of antibodies becomes unnecessary, thus eliminating the problemof reduced antibody activity often encountered. See, e.g. E. F. Ullmanand P. L. Khanna, supra. Third, the doubly-labeled peptide has a verylong Stroke's shift. If an argon ion laser (488 nm line) is used as theexcitation source, the fluorescence emission can be measured at 576 nm,instead of 515 nm. The Stoke's shift of 90 nm helps to avoidinterference from scattering and visible serum fluorescence at 500-515nm. Fourth, since the fluorescein fluorescence (515 nm) remains constantafter binding, it may be used as an internal self-reference point tocorrect for instrument fluctuations. Making use of this feature may alsoeliminate the need for standard curves in automated instruments. Fifth,in evanescent immunosensors, interference from bulk fluorescence isoften a serious problem. See, e.g. V. Hlady et al., Surface andInterfacial Aspects of Biomedical Polymers: Protein Adsorption, J. D.Andrade, Ed. (Plenum Press, New York, 1985), pp. 81-119. If thedoubly-labeled peptide is used as a tracer, it is fluorescent only whenbound to immobilized antibodies and would become non-fluorescent whendisplaced into bulk solution, thus there should be no problem of bulkfluorescence. This would significantly reduce the stringency requiredfor the optic detection system. Sixth, different fluorescent dye pairsmay be labeled onto different tracer peptides, thus proving thepossibility of detecting several analytes in the same samplesimultaneously. Seventh, this concept may also be applied to thehomogeneous detection of polynucleotides hybridization using a DNA probelabeled with two fluorophores.

The present invention is further illustrated with the followingExamples:

EXAMPLE I

The hCG epitope peptide and its conjugate with dyes. A peptide fromhuman chorionic gonadotrophin (hCG) was used as the spacer betweenfluorescein (F) and tetramethylrhodamine (T). Depicted in FIG. 1 is thestructure of a hCG epitope peptide labeled with fluorescein and TMR. Thepeptide was identified by screening 221 (24) overlapping octapeptidessynthesized on the tips of 96-pin solid supports as described in H. M.Geysen et al., Molecular Immunology, 23:709-715 (1986).

According to the Geysen method, a series of n-8 overlapping octapeptideswere synthesized on the tips of 96-pin solid supports and tested forspecific binding with anti-hCG using a ELISA procedure. where n is thenumber of amino acid residues in a sequence. A total number of 221octapeptides was screened because hCG has two chains and a total numberof 237 amino acid residues: R. B. Carlsen et al., J. Biol. Chem.,248:6810-6827 (1973); R. Bellisario et al., J. Biol. Chem.,248:6796-6809 (1973).

A core sequence of RLPGPSD (SEQ ID NO:1) showed strong reaction with theanti-hCG Mab. In order to conjugate dyes to the peptide withoutcompromising its binding properties, the sequence GSGSRLPGPSDTC (SEQ IDNO:2) was synthesized using standard Fmoc methodology (J. M. Stewart andJ. D. Young, Eds., Solid Phase Peptide Synthesis (Pierce, Rockford,Ill., 1984)) and purified on reversed phase HPLC to >95% purity. Itschemical identity was confirmed by Fab mass spectra and amino acidanalysis. The peptide was first reacted with TMR maleimide in 50 mM, pH6 phosphate buffer for 48 hours to make oligopeptide labeled with TMR(“pepT”). All chemically reactive dyes used were purchased fromMolecular Probes, Eugene, Oreg. After purification on reversed phaseFPLC (C-18 column, particle size 15 mm, Pharmacia LKB), pepT was reactedwith fluorescein succimidyl esters in 50 mM borate buffer, pH 8.5 forovernight to make FpepT. The dye-peptide conjugate has the chemicalstructure of F-GSGSRLPGPSDTC-T (FpepT) and is shown in FIG. 1. Agradient of acetonitrile in water was used to purify these conjugates.In a typical elution, the acetonitrile content was increased from 15% to30% over a period of 20 minutes, followed by an isocratic elution at 30%acetonitrile. All solvents contained 0.1% trifluoroacetic acid. Themolecular weight of FpepT measured by Fab mass spectra was 209T which is18 mass unit higher than the expected value of 2073.19. Thehydrophilicity of the peptide plus the bulky fluorophores probably havecaused entrapment of a bonded water molecule inside the conjugate.

Except for Cys, the linker peptide sequence corresponds to a portion ofthe naturally occurring sequence near the C-terminus of hCG b chain.This sequence, rather than the core peptide, was used because the latterexhibited little affinity to the anti-hCG Mab after fluorescein isattached at its N-terminus.

Comparison of the absorption spectra of FpepT, Fpep, and pepT showedthat the major absorption peak of fluorescein blue-shifted by 2 nm,while that of rhodamine red-shifted by 9 nm. Also, the long wavelengthpeak of rhodamine had actually become hypochromic by a factor of 1.6.

There are normally two electronic transitions in the visible absorptionregion of fluoresceins and rhodamines: upper and lower energy levels.The absorption spectrum of a monomer consists of an intense band at thelonger wavelength and a shoulder at the short wavelength. The formationof dimers provides a “flip-flop” of these relative peak intensities sothat the shorter wavelength transition is more hyperchromic. The changesin these two excited levels of the dimer have significant consequenceson its fluorescence spectra. The hypochromativity of the longerwavelength transition suggests a relatively long lifetime of the lowerenergy level. Due to radiation-less transitions from the upper to thelower excited level, most of the dimer molecules are in the lower statewhich exhibits weak fluorescence emission. Therefore, thisradiation-less relaxation process substantially contributes to the lowfluorescence quantum yield of the dimer.

Because of these changes, the absorption spectra of FpepT exhibited apoor fit to the simple sum of those of Fpep and pepT (FIG. 2),suggesting that ground-state static interactions exist between F and T.These static interactions have significant effect on the fluorescencequenching.

FIG. 2 graphically depicts the absorption spectra of the FpepT conjugate(solid line). The dashed line shows a least-squares fit of the spectrawith a model assuming a simple mixture of Fpep and pepT according to themethod of Wei, et al. Biosensor Design and Application, P. R. Mathewsonand J. W. Finley, Eds. (American Chemical Society, Washington, D.C.,1992), vol. 511, pp. 105-120. The residual between the measured valueand the fitted value at each wavelength is given in the inset. Because aperfect fit would generate a random residual plot with the average beingzero, the systematic residual pattern in this figure indicatessignificant ground-state interactions between the two dyes in the samemolecule. The concentration of this conjugate was deterrined by itsabsorbance at 560 nm using the molar extinction coefficientε_(560nm)^(1M)

of 60,000 M⁻¹cm⁻¹. The buffer system used for this spectra and an othersubsequent fluorescent studies was 100 mM phosphate buffer, pH 7.4,unless otherwise stated.

FIG. 3 depicts the technical fluorescence spectra of Fpep (1), pepT (2)and FpepT (3) when excited at 493 nm (c.a. 10⁻⁷ M). Compared to Fpep,the fluorescein fluorescence is quenched by 98% in FpepT.

FIG. 4 depicts the technical fluorescence spectra of pepT (1) and FpepT(2) when excited at 550 nm (10⁻⁷ M). Compared to pepT, the rhodaminefluorescence is quenched by 90% in FpepT. All fluorescence measurements(spectra, intensity, and polarization) were made with an ISS PC-1fluorometer (ISS, Champaign, Ill.) at 6° C. unless otherwise indicated.

FIG. 3 shows that the intrinsic fluorescence of rhodamine in the FpepTconjugate is 10 fold lower than in the absence of F (i.e., 90%quenching) as a result of contacting with fluorescein. The fluoresceinfluorescence of FpepT, on the other hand, is 64 fold lower than in theabsence of T (i.e., 98% quenching). The higher quenching efficiency ofthe fluorescein fluorescence results from both the static interactionwith and excited-stated energy transfer to rhodamine. These resultsstrongly suggest that free FpepT indeed exists as intramolecular dimers.

EXAMPLE II

Binding of FpepT with anti-hCG Mab. The fluorescence spectra of FpepTupon binding to the anti-hCG Mab is presented in FIG. 5. Thefluorescence of rhodamine (lambda max=570 nm) increased up to 5 foldwith increasing antibody concentration as a result of diminishedinteractions with fluorescein. The fluorescein fluorescence (lambdamax=515 nm), on the other hand, remained constant and quenched becauseof the fluorescence energy transfer from F to T. Since the distance atwhich 50% energy transfer efficiency occurs is 54 Å for the F-T pair,even if the peptide became fully extended after binding, the end-to-enddistance of 47.19 Å would still allow 70% energy transfer efficiency.For this reason, the fluorescein fluorescence was not enhanced althoughthe static quenching should be equally reduced for fluorescein as forrhodamine. The reduced ground state interaction between the two dyes canalso be seen by comparing the absorption spectra of FpepT in thepresence and absence of antibody.

FIG. 6 compares the absorption spectra of FpepT (6.5×10⁻⁶ M) in theabsence (dashed) and presence (solid) of anti-hCG antibodies (7.5×10⁻⁶M). After binding, the major absorption peaks of fluorescein andrhodamine have shifted more towards each other. As a result, the spectracan be better fitted with a model assuming a simple mixture of Fpep andpepT as shown in the inset. A.-P. Wei et al., in Biosensor Design andApplication, P. R. Mathewson and J. W. Finley, Eds. (American ChemicalSociety, Washington, D.C., 1992), vol. 511, pp. 105-120. An anti-hCGmonoclonal antibody from Organon Teknika, Boxtel, the Netherlands wasused. It had been prepared according to the procedure of R. v. Erp etal., J. Immunol. Methods, 140:235-241 (1991). Antibody concentration wasdetermined from its absorption at 278 nm, using an extinctioncoefficient of 14. Mouse immunoglobulin and bovine serum albumin werepurchased from Sigma Chemical Co. (St. Louis, Mo.).

As shown in FIG. 6, the major absorption peaks of fluorescein andrhodamine have shifted toward each other after binding. And, theabsorptivity of the rhodamine moiety has increased by a factor of 1.6because of dimer dissociation. As a result, the spectra could be betterapproximated by a simple mixture of the two respective dyes (FIG. 6,inset), indicating that the bound form of FpepT has less spectralperturbation than the free species. It should be noted, however, thatthe measured spectra of the bound species do not completely coincidewith the fitted spectra due to residual spectral perturbations thatstill exist.

A typical intensity-versus-antibody concentration profile for thebinding of FpepT with anti-hCG Mab is presented in FIG. 7. FIG. 7depicts graphically the fluorescence enhancement factor E as a functionof antibody concentration (EX=493 nm and EM=590 nm). Fluorescenceintensities of FpepT (1.3×10⁻⁷ M) in sample (Is, filled circle) andreference (Ir, open circle) cuvettes as aliquots of stock anti-hCG andBSA & mouse IgG solutions were added are shown in the inset. The valueof E was calculated from. $E = \frac{{Is} - {Ir}}{Ir}$

The E˜Po curve was fitted with the following equation using Kaleidagraph(Abelbeck Software):${2{Po}} = {\frac{KdE}{{Em} - E} + \frac{LoE}{Em}}$

where Po, Lo, Em and Kd are the total antibody concentration, totalFpepT concentration, maximum enhancement, and dissociation constant,respectively. The values of Kd and Em were found to be Kd=2.2×10⁻⁷ M andEm=4.1, respectively. A higher value of Em (Em=6.8) was obtained forEX=561 nm and EM=590 nm.

While the addition of anti-hCG resulted in gradual increase influorescence, the same amount of bovine serum albumin and nonspecificmouse IgG did not have any effect on the fluorescence of FpepT,indicating that the enhanced fluorescence is a result of specificbinding. The fluorescence enhancement factor (E) as a function ofantibody concentration (Po) was fitted with a classical binding equation(FIG. 7, inset). The maximum enhancement (Em) at 590 nm was found to be6.8 (ex=561), 4.1 (ex=493, data not shown), respectively. Thedissociation constant (Kd) was (2.2±0.3)×10⁻⁷ M (N=6). When the samepeptide was studied in the absence of fluorescein (i. e. with onlyrhodamine at the Cys position), the value of Kd was found to be0.67×10⁻⁷ M. The three-fold decrease in binding affinity after labelingwith fluorescein was probably caused by either steric hindrance orconformational differences between these two labeled forms of thepeptide.

Compared to FIGS. 3 & 4, Em values of 4.1 and 6.8 should correspond to51% and 78% of the intrinsic fluorescence of TMR, respectively. Resultsof control experiments confirmed this prediction. In a separate bindingexperiment, an antibody solution of fixed concentration was titratedwith aliquots of FpepT. The curve of E vs. FpepT concentration (Lo) wasfitted with a classical binding equation (FIG. 8).

FIG. 8. graphically depicts the fluorescence intensities as a functionof FpepT concentration (EX=561 nm and EM=590 nm). Sample cuvettecontained 1.4×10⁻⁷ M anti-hCG, and the reference cuvette contained1.5×10⁻⁵ M of BSA and mouse IgG. The Lo˜E curve was fitted with thebinding equation using Kaleidagraph (inset):${Lo} = {{2{Po}\frac{Em}{E}} - \frac{KdEm}{{Em} - E}}$

The value of Kd was (2.1±0.4)×10⁻⁷ M (N=3), in excellent agreement withthe result of FIG. 7.

EXAMPLE III

Binding specificity and reversibility. Aliquots of hCG were added to amixture of FpepT and anti-hCG to displace FpepT from the antibody. Asthe hCG concentration was increased, a series of spectra similar to FIG.5 but in reverse order were obtained, indicating the bound fraction ofFpepT was decreased (spectra not shown).

FIG. 9. graphically depicts the exchange of FpepT by hCG (EX=561 nm andEM=590 nm). A mixture of FpepT (5.5×10⁻⁸ M) and anti-hCG Mab (4.5×10⁻⁸M) was first prepared, and 5 μl aliquots of hCG stock solution (1100IU/ml) were added to the mixture. The reference cuvett contained onlyFpepT without antibody. The decrease in fluorescence intensity (inset)is due to displacement of FpepT by hCG. The fraction of bound FpepT(f2=E/Em) and the total hCG concentration (L^(o)1) follows the equation:${L^{o}1} = {\left\lbrack {\frac{{K1}\left( {{Em} - E} \right)}{K2E} + 1} \right\rbrack \left\lbrack {{2{Po}} - \frac{K2E}{{Em} - E} - {\left( {L^{o}2} \right)\frac{E}{Em}}} \right\rbrack}$

where K1, K2, Po, and (L^(o)2) are the disassociation constants of hCGand PpepT, total antibody concentration, and total FpepT concentration,respectively. The data set of E vs. hCG(l^(o)l) was fitted with theabove model. The values of K1, K2 were found to be 4.9×10⁻¹⁰ M and2.4×10⁻⁷ M, respectively. Human chorionic gonadotrophin (hCG) was a giftfrom Organon Teknika, Boxtel, the Netherlands. The concentration of hCGwas converted from IU/ml to mole/l using a specific activity of 11,200IU/mg for highly purified hCG and a molecular weight of 38,000.

FIG. 9 shows the fluorescence intensity as a function of concentrationsof hCG, BSA & mouse IgG. The decrease in fluorescence at 590 nm is onlyspecific to hCG, and little change was brought about by the non-specificproteins. These results confirmed the expectation that FpepT bindsspecifically to the same active site as hCG. The enhancement factor (E)vs. hCG concentration was fitted with a binding equation. The values ofKd for hCG and FpepT were found to be 4.9×10⁻¹⁰ M and 2.4×10⁻⁷ M,respectively. In spite of the 490-fold lower affinity exhibited by thetracer, the lowest detection limit of hCG was about 1×10⁻⁹ M. Thisexcellent assay performance is attributed to the fluorogenic propertieswhich allow preferential measurements of the bound tracer.

EXAMPLE IV

Fluorescence anisotropy. The anisotropy of rhodamine when excited at 550nm as a function of anti-hCG concentration is shown in FIG. 10. In FIG.7, (for excitation at 550 nm) anisotropies at 590 nm as a function ofconcentrations of anti-hCG and BSA & IgG are shown as filled and opencircles, respectively. The error bars represent the standard deviationsof three different experiments. The free FpepT has an anisotropy valueof Af=0.1087±0.0014 (N=17). The anisotropy value for bound FpepT isAb=0.3444. Similar results were obtained for ex=490 nm and em=515 nm(not shown). For excitation at 490 nm, anisotropies at 590 nm as afunction of concentrations of anti-hCG and BSA & IgG are shown as filledand open squares, respectively. This property can be used for afluorescence polarization assay.

The more than 3-fold increase in anisotropy was attributed to the largedifference in size between FpepT (MW=ca. 2000) and the antibody (MW=ca.150,000). Similar result was obtained for the anisotropy of fluoresceinwhen excited at 490 nm (data not shown). Because the emission was fromthe low-energy absorption band in these measurements, the limitinganisotropy value is expected to be in the range of 0.39-0.4 for bothfluorophores. If the rotational diffusion of the intact IgG and the Fabfragments is taken into account, the observed maximum anisotropy valueof 0.3444 for the bound FpepT suggests that both fluorophores havelittle rotational mobility in the antibody-FpepT complex, because of thesmall sized antibody active site and the bulky fluorophores.

When, however, the excitation was 490 nm and fluorescence was measuredat the rhodamine emission, a completely different trend of change inanisotropy was obtained. Under this condition, because the high-energyabsorption band was excited, the anisotropy is more a measure of theangle between the absorption and emission dipoles. The anisotropy of thefree FpepT was 0.0436±0.0023 (N=17), as compared to 0.1087±0.0014 (N=17)when excited at 550 nm. The depolarization effect is caused by thenon-colinearity between the absorption dipole at 490 nm and the emissiondipole. As the anti-hCG concentration was increased, if there were nochange in the angle between these two dipoles, an increase in anisotropydue to the diminished rotational motion would be expected. However, thedata shows that increasing amount of antibody actually results in agradual decrease in the anisotropy which saturates at near zero. Thisphenomenon strongly indicates that the angle between the two dipoles ofFpepT had actually become larger when bound.

These results, together with those depicted in FIGS. 5 and 6, supportthe conclusion that the observed fluorescence enhancement was indeed dueto the dissociation of the intramolecular dimers between fluorescein andrhodamine as a result of conformational changes in FpepT.

EXAMPLE V

Fluorescein/anti-fluorescein as a model system. In addition to thehCG/anti-hCG system, a model system was also studied which consists ofan anti-fluorescein Mab (4-4-20) and an antigen made of fluoresceinlinked to rhodamine via a hexane spacer (i.e. FhexT). The structure ofthis conjugate is shown in FIG. 11. To make FhexT, diaminohexane wasreacted with an equal molar mixture of 5-isothiocynates of fluoresceinand TMR in 100 mM carbonate buffer, pH 9.5 for overnight. The FhexTconjugate was isolated from the reaction mixture on reversed phase FPLCusing the same condition as in FIG. 2. The absorption spectra of thisconjugate is shown in FIG. 12. The bands at 515 mn and 555 nm arecharacteristic of the spectral splitting due to ring stacking asobserved for rhodamine homo-dimers. I. L. Arbeloa and P. R. Ojeda,Chemical Physics Letters 87:556-560 (1982).

Concentrations of the conjugates used in FIG. 12 were determined fromthe absorbance at 556 nm using the extinction coefficient of 58,000 M−1cm−1. In this particular case, however, the ring stacking is due to theformation of fluorescein-rhodamine hetero-dimers. Another indication ofstacked dimer formation was the low level of fluorescence observed forFhexT. The fluorescence of free fluorescein or rhodarine at the sameconcentration would be about 1000-fold higher than that of theconjugate. As shown in FIG. 13, the fluorescence of rhodamine (lambdamax=576 nm) increased with increasing concentrations of (Fab′)2, anantigen-binding fragment of 4-4-20, while that of fluorescein (lambdamax=515 nm) remains at a constant low level. This observation isconsistent with the results of FIGS. 5 and 6, except that theenhancement in this case is about 400-fold, rather than 5-fold. Thisdifference is attributed to two factors. First, the close distancebetween the two dyes in FhexT (˜10 Å) allows almost 100% energy transferefficiency from fluorescein to rhodamine. It also facilitates stackeddimer formation which quenches fluorescence more efficiently. Second,because fluorescein is the antigen in this system, it fits tightly intothe binding site of the 4-4-20 Mab. J. N. Herron et al., Proteins:Structure, Function, and Genetics, 5:271-280 (1989). The staticinteraction with rhodamine after binding is thus diminished moreeffectively in this system than in the hCG/anti-hCG system. For thesereasons, much higher enhancement of rhodamine fluorescence was observed.Binding specificity and reversibility of FhexT with 4-4-20 (Fab′)2 wasstudied using 5-aminofluorescein are a nonfluorescent derivative offluorescein. J. N. Herron, in Fluorescein Hapten: An ImmunologicalProbe, J. E. W. Voss, Eds. (CRC Press, 1981) pp. 53-55.

The FhexT-antibody complex from the above titration experiments (FIG.12) were titrated with 10 ml aliquots of 5-aminofluorescein (10−4M) inorder to exchange the FhexT. Measurements were made after stirring for 2minutes in the cuvette holder at 25° C. Spectra measured after 2 minutesand 30 minutes were found to be completely superimposable, indicatingthat binding equilibrium was achieved in a period of less than 2 min.

FIG. 13 is the technical fluorescence spectra of the FhexT conjugate(4.35×10⁻⁷ M) as aliquots of (Fab′)2 fragment of antifluorescein 4-4-20were added. The total (Fab′)2 concentration changed from zero to2.6×10⁻⁷ M with a dilution factor of 3.6%. The concentration of (Fab′)2was determined by absorbance at 278 nm using an extinction coefficientof 1.5 and molecular weight of 110,000 daltons.

A series of spectra similar to FIG. 13 but in reverse order wereobtained as increasing amount of aminofluorescein was added, resulting atypical exchange curve (data not shown). BSA was found to have no effecton the fluorescence intensity of FhexT in either bound or unbound state.These results indicate that the FhexT tracer binds to 4-4-20specifically and reversibly. Although the PhexT/4-4-20 system needs tobe further characterized to obtain biophysical parameters, it certainlyserves as a template for optimizing the FpepT tracer or developing othertracers because of the efficient quenching and dramatic enhancement offluorescence it exhibited.

Generality and fluorophore selection. The hCG/anti-hCG system describedherein was completely a random choice. From this stand-point, theseresults should be of general applicability. Although the hCG epitope wasidentified based upon the hCG sequence, recent peptide technologies havemade it possible to identify a high affinity antigen-mimicking peptidefor any monoclonal antibody. It therefore follows that the inventiveapproach may also be applied to protein antigens of unknown sequence orother non-protein antigens. Proper labeling of the peptide withfluorophores is the most critical aspect of these fluorogenic tracers.Although the core peptide is usually 6 to 8 amino residues in length, alonger sequence was used in order to reduce steric hindrance. The lengthof peptide will, in turn, determine what fluorophores to use. Assuming atypical dimerization constant of 2,500 M−1, the concentration offluorescent dyes required to form 90% dimers is 1.8×10⁻² M which is1×1019 molecules/cm3. Under this condition, the average distance betweentwo dye molecules is about 60 Å (measured from the centers of mass).Since the extended distance between alpha-carbons in polypeptide is 3.63Å, the distance of 60 Å corresponds to about 16 amino acid residues. Inother words, if the dye has a Kd of 2,500 M−1, the two fluorophores inthe tracer can be spaced, at most, by 16 residues. This provides thebasis for selecting fluorophores for a given peptide length. If thepeptide is shorter than a 16-mer, fluorophores with smaller Kd should beused, and the vice versa, in order to achieve effective intramoleculardimerization. The dimerization constants of fluorescein, eosin,rhodamine B, rhodamine 6G are 5, 110, 2100, 5600 M−1, respectively (22,33, 34), while that of cyanines varies in the range of 103-106 M−1depending upon the chain length of the alkyl linkers in theirstructures. W. West and S. Pearce, J. Phys. Chem., 69:1894-1903 (1965).

EXAMPLE VI

Materials and Methods

Chemicals and Reagents

The purified 5- and 6-isomers of fluorescein isothiocyanate andtetramethylrhodamine isothiocyanate, 5-(and-6) carboxyfluoresceinsuccinimidyl ester, 5-(and-6) carboxytetramethylrhodamine succinimidylester were products of Molecular Probes (Eugene, Oreg.).Ethylenediamine, 1,4-butanediamine, 1,6-hexanediamine,1,8-octanediamine, 5-aminofluorescein (isomer I) were purchased fromAldrich Chemical Company (Milwaukee, Wis.). All solutions were made in100 mM phosphate buffer (pH 7.4) unless otherwise indicated.

Preparation of Antifluorescein Mab and Its Fragments

Antifluorescein monoclonal antibodies (Mabs) 4-4-20 and 9-40 weregenerated through chemically mediated fusion of BALB/c spleniclymphocytes with the Sp 2/0-Ag 14 myeloma cell line. Hybridoma celllines were obtained from Prof. E. W. Voss, Jr. at University of Illinoisat Urbana-Champaign. Kranz et al. Mol. Immunol., 18:889-898 (1981);Bates et al. Mol. Immunol., 22:871-877 (1985). Mabs were purified frommouse ascites fluid. Ammonium sulfate precipitation was followed byDEAE-cellulose anion exchange and chromatofocusing FPLC (Pharmacia) overa pH gradient of 7.0 to 5.0. Preparations were characterized by gelelectrophoresis and fluorescence quenching assays. Herron, J. N. InFluoroscein Hupten: An Immunological Probe, E. Voss Jr. Ed. (1981 CRCPress, Boca Raton, Fla.) pp. 53-55. Antibody solutions were filteredthrough Durapore 0.2 μm filters (Mipore, Bedford, Mass.) before use. Themolar concentration of (Fab′)₂ and intact antibody was determined byabsorbance at 278 nm using an extinction coefficient (ε_(1cm) ^(1%)) of14 and molecular weight of 110 and 150 kDa, respectively,

Preparation of F—(CH₂)_(n)—T Bichromophores

Single-labeled conjugates were first prepared by reactingfluorescein-5-isothiocyanate (˜5 mg) with ca. a 10-fold molar excess ofeither 1,2-ethylenediamine, 1,4-butanediamine, 1,6-hexanedianine, or1,8-octanediamine in 100 mM carbonate buffer (pH 9.5) overnight at roomtemperature. The single labeled conjugates (F—(CH₂)_(n)—NH₂) wereseparated from reactants and other products using reversed phase FPLC(C-18 column, particle size 15 μm, Pharmacia LKB). Each reaction mixturewas eluted at a flow rate of 1 ml/min with a linear gradient ofdeionized water (H₂O) and acetonitrile (ACN) both of which contain 0.1%trifluoroacetic acid (TFEA). For a typical elution, ACN was increasedfrom 15% to 50% in a 45-minute interval followed by a 15-min isocraticelution at 50% ACN. In the second step, purified fractions ofF—(CH₂)_(n)—NH₂ were reacted with excesstetramethyfrhodamme-5-isothiocyanate (˜1.5-fold), respectively to makebichromophores. The reaction and purification conditions were the sameas in the first step. In order to ensure purity, each of the F—(CH₂)₂—T,F—(CH₂)₄—T, F—(CH₂)₆—T, F—(CH₂)₈—T preparations were repurified using 50mM phosphate buffer (pH 7.4), instead of deionized water, as the aqueousphase during the elution gradient. The chemical identity of thesecompounds was confirmed by fast atom bombardment (FAB) or electrospray(ES) mass spectrometry. The concentration of these bichromophores weredetermined from the rhodamine absorption maxima at ˜560 nm using anextinction coefficient of 37,500 M⁻¹ cm¹. Similar reaction andseparation conditions were used to prepare F—(CH₂)₆—T conjugatesinvolving 6-isomers and amide linkages.

Spectral Analysis

Visible absorption spectra of F—(CH₂)_(n)—T in the absence and presenceof 4-4-20 were subjected to multiple linear regression analysis usingthe model:

A _(F—(CH2)n—T) =α·A _(F—(CH2)6) +β·A _(T—(CH2)5)+ε  (1)

where A_(F—(CH2)6), A_(T—(CH2)5), and A_(F—(CH2)n—T) are the absorptionspectra (400-650 nm) of single-labeled and double-labeled conjugates,respectively; α and β are linear coefficients to be determined; and ε isthe residual term. The analysis was performed on an Apple Macintoshcomputer using StatWorks (Cricket Software Inc., Philadelphia, Pa.). Allabsorption spectra were measured on a Perkin-Elmer Lambda 2 UV/Visspectrometer at room temperature (˜25° C.)

Fluorescence Measurements

Fluorescence spectra, intensity, and anisotropy measurements were takenwith an ISS PC-1 fluorometer (ISS, Champaign, Ill.). An excitationwavelength of 488 (fwhm dispersion=4 nm) was used, and fluorescenceemission was measured through a 589 nm interference filter (fwhm=10 nm,Oriel, Conn.) superimposed with a 570 nm long pass filter (Schott, Pa.).Temperature was controlled at 25° C. using a water bath. In alltitration experiments, the overall titrating volume added to the samplewas less than 4% of the total sample volume.

Binding Experiments and Data Analysis

For each conjugate, two identical solutions of F—(CH₂)_(n)—T (˜1×10⁻⁸ M)were prepared. One was titrated with 5 μL aliquots of a stock 4-4-20solution (sample), and the other with a 1:1 mixture of BSA and mouse IgG(reference). Fluorescence intensities in sample and reference cuvetteswere denoted as I_(s) and I_(r), respectively. The background intensity(I_(b)) was measured before any conjugate and antibody were added. Theenhancement factor is defined as: $\begin{matrix}{E = \frac{I_{s} - I_{r}}{I_{r} - I_{b}}} & (2)\end{matrix}$

Because these conjugates are nearly non-fluorescent (when not bound),the denominator term (I_(r)−I_(b)) is often very small. Any smallvariation in it could cause large changes in the value of E. In order toavoid this problem, we adopted a different form of this equation asshown below: $\begin{matrix}{E = {\left( \frac{I_{s} - I_{r}}{I_{r}} \right)\left( \frac{I_{r}}{I_{r} - I_{b}} \right)}} & (3)\end{matrix}$

Mathematically, this expression is equivalent to eq 2. It can be shownthat E and the total antibody concentration (P_(o)) are related asfollows: $\begin{matrix}{{{2P_{o}} = {\frac{K_{d}E}{E_{m} - E} + \frac{L_{o}E}{E_{m}}}},} & (4)\end{matrix}$

where P_(o), L_(o), E_(m) and K_(d) are the total antibodyconcentration, total F—(CH₂)_(n)—T concentration, maximum enhancement,and dissociation constant, respectively. The E vs P_(o) data set was fitto this equation using Kaleidagraph (Abelbeck Software). This procedureof determining binding parameters is referred to as method I insubsequent discussions. In an alternate method (method II), a samplesolution of 4-4-20 (1×10⁻⁸ M) was titrated with 5 μL aliquots of stockF—(CH₂)_(n)—T. The same amount of F—(CH₂)_(n)—T was also added to areference buffer solution. The relationship between total F—(CH₂)_(n)—Tconcentration (L_(o)) and E is given by the equation: $\begin{matrix}{{L_{o} = {{2P_{o}\frac{E_{m}}{E}} - \frac{K_{d}E_{m}}{E_{m} - E}}},} & (5)\end{matrix}$

where the parameters are defined the same as in eq 3. The E vs L_(o)data set was fit to this equation using Kaleidagraph. Eqs 4-5 werederived from the basic mass law of binding equilibrium. Readers shouldrefer to Herron (1981) supra or Pesce et al. for general derivationprocedures. Pesce et al. Flourescence Spectroscopy: An Introduction forBiology and Medicine (Marcel Dekker NY 1971).

Results and Discussion

The Bichromophoric Conjugates

Six bichromophoric conjugates were studied. Their structures are shownin FIG. 16. They consist of fluorescein (F) linked totetramethylrhodamine (T) by varying numbers of CH₂ units throughthiourea bonds or amide bonds. A conjugate using the 6-isomers of F andT was also studied for the effect of isomeric states. These compoundswere purified to >98% purity on reversed phased FPLC and stored at −20°C. Their chemical identity was confirmed by mass spectrometry theresults of which are shown in Table 1. Because the thiourea bonds arerelatively labile, chemical decomposition is possible after long-timestorage, resulting in substantial increase in background fluorescence.In order to avoid this problem, conjugates were usually measured withinthree days of fresh preparation. If storage was beyond three days, theywere repurified on an FPLC column before use.

The F—(CH₂)_(n)—T conjugates were first examined for ground-stateinteractions between F and T. As an example, the absorption spectrum ofF—(CH₂)₆—T (III) is shown in FIG. 17. It has two major electronictransitions in the visible region—491 and 563 nm—representing dipoles offluorescein and tetramethylrhodamine, respectively. This spectrum wasfit to eq 1 by a multiple linear regression procedure. Compared to thesummed spectra of single-labeled conjugates, the absorption maximum offluorescein is blue-shifted by 2 nm, while that of rhodamine isred-shifted by 11 nm. Similar results were also obtained for compoundswith n=2, 4, 8 (data not shown), indicating that significant groundstate interactions indeed exist between F and T in these conjugates.

TABLE 1 Description of six compounds shown in FIG. 16 Compound I II IIIIV V VI Linker Length n = 2 n = 4 n = 6 n = 8 n = 6 n = 6 Chemistrythiourea thiourea thiourea thiourea thiourea amide Isomeric State 5, 55, 5 5, 5 5, 5 6, 6 5, 5 Mol. Mass (C) 893.00 921.05 949.11 977.16949.11 886.96 Mol. Mass (M) 892.84 920.84 948.84 976.84 948.84 886 C -Calculated mass; M - Measured by FAB or Electrospray mass spectrometry.

A more dramatic change in absorption spectra was observed for F—(CH₂)₆—T(V) which contains 6-isomers of F and T. As shown in FIG. 18, theabsorbance consists of three separate transitions. The peak at 490 nm isattributed to fluorescein. Peaks at 515 nm and 555 nm are attributed torhodamine in hetero-dimeric states with fluorescein. Normally, theabsorption of rhodamine molecules consists of a major transition at 550nm and a shoulder at about 30 nm to the blue. In monomeric states, thelonger wavelength transition is the stronger of the two. However,dimerization provides a “flip-flop” of these relative peak intensitiesso that the shorter wavelength transition is more hyperchromic. Theexistence of these two excited levels of the dimer has significantconsequences on its fluorescence quantum yield. The hypochromativity ofthe longer wavelength transition suggests weak oscillator strength and arelatively long lifetime of the lower energy level. Due to radiationlesstransitions from the upper to the lower excited level of the dimer, mostof the dimer molecules may be in the lower state which exhibits weakfluorescence emission. Therefore, this radiationless relaxation processmay substantially contribute to the low fluorescence quantum yield ofthe dimer. For this reason, these conjugates are nearly non-fluorescent(>99% quenching).

Binding of F—(CH₂)₆—T (V) with Antifluorescein 4-4-20

As shown in FIG. 19, the fluorescence of F—(CH₂)₆—T increased with theconcentration of (Fab′)₂—a bivalent antigen-binding fragment. This is aresult of specific binding between the antibody and F—(CH₂)₆—T.Antifluorescein 4-4-20 is a high affinity antibody for fluorescein(K_(d)˜10¹⁰ M). When it binds to fluorescein in the F—(CH₂)₆—Tconjugate, the intramolecular monomer⇄dimer equilibrium is driventowards the monomer side because the active site can not accommodateboth fluorophores. This structural change causes significant increase influorescence intensity because of the intramolecular dimer dissociation.It is important to note, however, that only the fluorescence ofrhodamine (λ_(max)=576 nm) is enhanced, and that of fluorescein(λ_(max)=515 nm) remains low and constant. This observation may beattributed to two factors. First is the resonance energy transfer (RET)from F to T. The efficiency of RET is proportional to the inverse 6thpower of the distance between donor and acceptor molecules. Because themaximal distance between F and T in the conjugate is ˜10 Å, the transferefficiency is almost 100%, suggesting that the excited-state energy of Fmay be completely transferred to T. Second is the high degree ofquenching in the antibody active site. It is well known that 4-4-20quenches fluorescein fluorescence by 96% upon binding due tointeractions with active site residues. These quenching effects forfluorescein are in many cases desirable because they produce a largeeffective Stoke's shift for the F—(CH₂)_(n)—T conjugates. The conjugatescan be excited at 488 nm (a common laser line) and emit at ˜λ_(max)=580nm. This property enables the rejection of background interference dueto light scattering or endogenous fluorescence in biological samples.

FIG. 20 is a plot of fluorescence enhancement (E), calculated from theintensity values at the emission maximum (575 nm), vs (Fab′)₂concentration (P_(o)) This data set was fit to eq 3. The maximumenhancement (E_(m)) was found to be 28.5 and the dissociation constant(K_(d)) was 6×10⁻¹⁰ M. The value of K_(d) is slightly higher than thatfor free fluorescein (˜10⁻¹⁰ M) because a small portion of the bindingfree energy was devoted to dissociate the F and T heterodimer which haveK_(d) values of ˜10⁻² to 10⁻³ M. It should be pointed out that themagnitude of fluorescence enhancement (˜30-fold) is exceptional andunparalleled by other comparable systems involving fluorescentimmunoreactions. In most RET-based methods reported in the literature,it is the donor fluorescence that is modulated, rather than that of theacceptor. Stryer et al. Proc. Natl. Acad. Sci. USA 58:719-726 (1967);Ullman et al. Methods in Enzymology, 74: 28-60 (1981). In some cases,the binding-mediated intensity changes are very marginal. Barnard et al.Science, 251: 927-929 (1991).

The reversibility of the antibody-mediated fluorescence enhancement wasexamined by titrating a mixture of 4-4-20 and F—(CH₂)₆—T with5-aminofluorescein which is a nonfluorescent analog of fluorescein.Spectra shown in FIG. 21 demonstrate that rhodamine fluorescence(λ_(max)=576 nm) decreases with increasing concentration of5-aminofluorescein. The slight increase at ˜515 nm is attributed to theresidual fluorescence of 5-aminofluorescein itself. This experimentindicates that F—(CH₂)₆—T can be displaced from antibody sites into itsquenched state again. BSA and mouse IgG had no effect on thefluorescence intensity of F—(CH₂)₆—T in either bound or unbound states.These results indicate that the fluorescence enhancement observed wasindeed specific and reversible.

Dependence on Linker Length

Four conjugates with n=2, 4, 6, 8 were studied in order to examine theeffect of linker length on the binding properties of F—(CH₂)_(n)—T. Inthe first experiment, F—(CH₂)_(n)—T solutions of fixed concentration(˜10⁻⁸M) were titrated with aliquots of stock 4-4-20 solution.Fluorescence intensities thus obtained were analyzed according to methodI described in Materials and Methods. FIG. 22 shows the enhancementfactor (E) as a function of antibody concentration for the fourconjugates. In the second experiment, 4-4-20 solutions of fixedconcentration (˜10⁻⁸ M) were titrated with aliquots of stockF—(CH₂)_(n)—T solution. Fluorescence intensities thus obtained wereanalyzed according to method II. FIG. 23 shows the enhancement factor(E) as a function of F—(CH₂)_(n)—T concentration for the fourconjugates. Values of dissociation constant (K_(d)) and maximumenhancement factor at 589 nm E_(m) ⁵⁸⁹ are summarized in Table 2 andplotted in FIG. 24. The results show that as the linker becomes longer,the binding affinity increases while the fluorescence enhancementdecreases.

TABLE 2 Summary of binding parameters obtained in FIG. 20 Dissociationconstant K_(d) (nM) Maximal Enhancement Factor (E_(m) ⁵⁸⁹) I II III IV III III IV Compound (n = 2) (n = 4) (n = 6) (n = 8) (n = 2) (n = 4) (n =6) (n = 8) Method I 1.3 3.4 1.0 0.6 114.6 108.4 41.8 26.2 Method II 5.02.6 0.7 N/A 109.9 95.5 42.1 N/A Average 3.2 3.0 0.9 0.6 112.3 102.0 42.026.2

The excited-state energy of one rhodarine molecule can be transferredefficiently to another at a proximity of <50 Å. This is a resonanceenergy transfer process that can occur through space without the needfor physical contact between the donor and acceptor molecules. Becauseboth factors are distance-dependent, a longer linker would have moreflexibility and can bring two rhodamine molecules closer together. Forthis reason, the fluorescence enhancement factor decreases as the linkergets longer. Apart from the above explanations, two other trivialfactors may also contribute to the observed linker effect on E_(m) ⁵⁸⁹.First, a longer linker has a higher degree of conformational freedomwhich may reduce the stacking efficiency between F and T. Because ofthis, the level of background fluorescence (I_(b)) may be elevated.According to the definition of E (eq 2), a higher I_(b) would correspondto a lower E_(m) ⁵⁸⁹. However, control experiments showed littledifference in background fluorescence among the four conjugates,indicating this is probably a trivial factor. Second, a longer linkermay allow rhodamine molecules to fold up and remain associated withfluorescein even when the latter is bound to its antibody. However,examination of the 3-dimensional structure of the 4-4-20 Fab-fluoresceincomplex reveals that the size, geometry and residue positioning of theactive site are all tailored to bind with fluorescein. Herron et al.Proteins: Structure, Function and Genetics, 5:271-280 (1989). It isunlikely that both fluorophores can be accommodated in the active site.Although dynamic interactions of rhodamine with neighboring residues mayquench some of its fluorescence, this is probably also a trivial factorbecause such dynamic quenching should occur for all linkers.

Effect of Linking Chemistry

All conjugates discussed so far employ isothiocyanate chemistry forlinkages between fluorophores and the alkyl chains. However, thioureabonds are known to be labile and can result in significant chemicaldislocation especially after long-term storage. For this reason, wedecided to introduce amide chemistry into one of the conjugates (n=6) toprepare F—(CH₂)₆—T (VI). Its spectral and binding properties werecompared to those of F—(CH₂)₆—T (III). Although the changes in chemistryare small, they do have profound effect on its properties. In the firstexperiment, fluorescence spectra of F—(CH₂)₆—T (VI) (3×10⁻⁷ M) weremeasured when aliquots of 4-4-20 solution were added. Results are shownin FIG. 25. When unbound, the conjugate has a predominant peak fromfluorescein and a shoulder peak due to rhodamine. Binding to 4-4-20provides a flip-flop in the relative intensities of F and T. Thedecrease in fluorescein fluorescence is attributed to binding-associatedquenching in the active site, while the increase in rhodaminefluorescence is a result of dissociation of the intramolecularhetero-dimers between F and T. These results indicate that bothconjugates exhibit similar properties in terms of binding-mediatedmodulation of rhodamine fluorescence. However, the amide chemistrycauses incomplete quenching of fluorescein fluorescence in the freestate, presumably, due to inefficient stacking and/or improperorientation alignment between the two fluorophores.

In the second experiment, we were interested in a more quantitativecomparison of differences in binding affinity and enhancement factors. Atypical titration curve was obtained by titrating a F—(CH₂)₆—T (VI)solution of fixed concentration (1.8×10⁻⁷ M) with aliquots of 4-4-20solution (FIG. 26). The E˜P_(o) data set was fit to eq 3, giving a valueof 5×10⁻⁷ M for K_(d) and 24.8 for E_(m) ⁵⁸⁹. The nearly 25-foldfluorescence enhancement was specific to 4-4-20 and can be reversed bythe addition of 5-aminofluorescein. This enhancement value is comparableto those obtained in FIG. 17 and FIG. 20, although slightly lower inmagnitude. A more significant difference is in the value of K_(d)—about500-fold lower than that shown in Table 2 (Kd=0.9 nM for n=6).Conjugates with the thiourea linkage can bind better because theirstructures more closely mimic the original immunogen in whichfluorescein was linked to the side chain of lysine residues of a carrierprotein via the thiourea bond. Although both conjugates VI and III havesix CH₂ units in their linker, VI is two bonds shorter than the latter(measured from 5C of F to 5C of T, see FIG. 15) because of , differentlinking chemistry. As discussed earlier, shorter linkers tend to reducebinding affinity because of steric hindrances. In addition, a morefundamental explanation may be found by examining the two linkages:

where Ar stands for an aromatic ring. In the amide bond, the Ar—C bondis rotable and O, C, N atoms are co-planar due to electrondelocalization. In the thiourea bond, however, there are no rotablebonds and Ar, N, C, S, N are all co-planar. Because intramoleculardimerlization requires F and T to fold upon each other, bond rotationand flexibility are necessary conditions for the formation of tightdimers. Therefore, we can reasonably conclude that F and T are moreefficiently stacked when amide chemistry is used. Supporting thisconclusion is the diminished affinity of F—(CH₂)₆—T (VI) with anotherantifluorescein antibody 9-40 which comes from the same gene family as4-4-20 but has lower affinity toward fluorescein (K_(a)˜10⁷ M⁻¹).

Polarization Spectra

The bright rhodamine fluorescence observed for the bound bichromophoresmay result from two mechanisms: direct excitation of the rhodamineoscillator at 488 nm after de-quenching by binding and/or resonanceenergy transfer (RET) from F to T. Although both mechanisms arepossible, RET probably dominates the process. Control experiments showedthat the fluorescence intensity of monomeric rhodamine is less than 10%of the level observed for the bound F—(CH₂)₆—T (VI) conjugate whenexcited at 488 nm. This indicates that because of the weak oscillatorstrength at this wavelength, direct excitation only accounts for a smallportion of the observed enhancement of rhodamine fluorescence. Themajority is contributed by the excited-state energy transfer process.This is best illustrated by the result in FIG. 27 which shows thefluorescence polarization spectra of free and bound F—(CH₂)₆—T (VI)conjugate in comparison with that of a rhodamine molecule bound to anantirhodamine antibody. The wavelength at which the major polarizationtransition occurs is shifted from ˜450 nm for the bound rhodatnine to˜525 nm for the bound F—(CH₂)₆—T (VI). This suggests that there is anadditional dipole which, although not intrinsically present in therhodamine molecule, can still relax from its excited state into thelowest singlet state of the rhodamine emission dipole. The new dipole isclearly introduced by the presence of fluorescein. This is, in fact, aconsequence of the Forster energy transfer theory. It should be notedthat the excitation dipole of fluorescein and the emission dipole ofrhodamine are not coplanar. Also, the fact that the anisotropy value ofthe bichromophore does not level off at the maximal value of 0.4 (asfound for antibody-bound rhodamine) suggests that there is significantdepolarization effect. This demonstrates that rhodamine has higherrotational freedom when linked to antibody-bound fluorescein than whenitself is bound to an antibody

In summary, we have demonstrated that the fluorescence emission oftetramethylrhodamine (T) can be modulated by antibodies that are highlyspecific to fluorescein (F) and do not cross-react with T. This isachieved by conjugating F and T via an oligomethylene spacer to make aso called bichromophore. Due to the short interchromophore distance, Fand T can fold upon each other to form stacked intramolecularhetero-dimers. As a result, both fluorophores are essentiallynon-fluorescent. However, when an antifluorescein antibody binds to thefluorescein moiety of the bichromophore, the dynamic monomerxdimerequilibrium is driven towards the monomeric form which is highlyfluorescent. Therefore, the fluorescence emission is effectively coupledto the antibody-ligand binding events. As an added advantage, thissystem makes use of the resonance energy transfer properties between Fand T so that the excited-state energy of fluorescein can be transferrednonradiatively to rhodamine which in turn emits its own fluorescence. Ineffect, the bichromophore is a molecule with a long Stoke's shift whosefluorescence emission can be modulated by binding to antibodies. Thiscombinatorial use of fluorescent dye dimerization and fluorescenceenergy transfer is novel and unique, especially in the context ofcoupling these phenomena to biomolecular binding events.

The implications of this study can be seen from several perspectives.First, antifluorescein antibodies quench ligand fluorescence uponbinding. This property provides a convenient means to measure bindingparameters in research laboratories. However, quenching is not alwaysdesirably in some experiments (e.g. polarization measurements) becausethe bound species contribute little to the total fluorescence intensity.This disadvantage can be offset by introducing fluorescence enhancementinto the system. Enhancement combined with the long Stoke's shift willalso help expand the applications of the antifluorescein system fromlaboratory use into practical areas such as biosensors, Wei et al.Biosensor Design and Application, 511:105-120 (American ChemicalSociety, Washington D.C. 1992) clinical immunoassays, Wei et al. Anal.Chem., 66:1500-1506 (1994) fluorescence activated cell sorters Karawajewet al. J. Immunol. Methods, 111:95-99 (1988) and characterization ofliposomal vesicles. Second, the concept described herein (see FIGS. 14and 15) is not limited to the antifluorescein model system. The factthat similar results were obtained with two different systems (e.g. hCGand antifluorescein) involving both high and low molecular antigenssuggest that the concept is quite general. This is important because ourapproach for the first time combines fluorescent detection withmolecular recognition—two events which were usually separated in mostother systems. Wei et al. (1992) supra. Third, the magnitude ofantibody-mediated fluorescence enhancement found in this study isphenomenal—up to 110-fold depending upon the linker length. This can beused as a model to improve other systems and to understand thefundamental biophysical mechanisms behind it.

EXAMPLE

A homogenous DNA hybridization assay in which a pair of fluorophores(fluorescein and rhodamine) is attached to the 5′ and 3′ ends,respectively, of an oligonucleotide (30 nucleotides in length) which iscomplementary to a target DNA sequence that is part of a much largerpiece of DNA (chromosomal DNA). The flourescently-labeledoligonucleotide is mixed with the target DNA and the mixture heated to atemperature high enough to denature the double helix. As the mixturecools, the oligonucleotide hybridizes with its target sequence. Theunbound form of the fluorescently labeled oligonucleotide isnon-fluorescent because of dimer formation between the two dyes. Uponhybridization however, this dimer dissociates, resulting in an increasein fluorescence. As such, this is a homogenous, solution phase assaybecause no wash steps are required. Its sensitivity would be limited,however, by the sensitivity of the fluorometer for bulk fluorescence(e.g. one picomolar would be a practical limit with currentinstrumentation).

EXAMPLE

The experiment of the previous EXAMPLE is repeated somewhat to produce asolution-phase homogenous RNA hybridization assay in which a pair offluorophores (Cy-3 and Cy-5) is attached to the 5′ and 3′ ends,respectively of an oligonucleotide (10 nucleotides in length) which iscomplementary to a target RNA sequence that is part of a larger piece ofRNA. The target RNA is denatured first, for example by heating it to atemperature high enough to denature the double helix, at which point theflourescently-labeled oligonucleotide is added and then mixed with thetarget RNA. As the mixture cools, the oligonucleotide would hybridizewith its target sequence. The unbound form of the fluorescently labeledoligonucleotide is non-fluorescent because of dimer formation betweenthe two dyes. Upon hybridization however, the dimer dissociates,resulting in an increase i fluorescence. As such, this too is ahomogenous, solution phase assay because no wash steps are required.

The EXAMPLES were provided to illustrate certain embodiments of thepresent invention and, for that reason, should not be construed in alimiting sense.

2 7 amino acids amino acid linear 1 Arg Leu Pro Gly Pro Ser Asp 1 5 13amino acids amino acid linear 2 Gly Ser Gly Ser Arg Leu Pro Gly Pro SerAsp Thr Cys 1 5 10

We claim:
 1. A procedure for detecting and quantifying a first DNA inbulk solution comprising: (a) placing in the bulk solution a conjugatecomprising: a second DNA strand complementary to at least a portion ofsaid first DNA, said second DNA strand having a pair of fluorescentmaterials capable of forming a dimer joined thereto with sufficientnucleotides between said pair of fluorescent materials so the second DNAstrand is capable of folding upon itself, said conjugate capable ofmoving between a more stacked configuration and a more stackedconfiguration to exhibit a change in fluorescence; and (b) adding thefirst DNA to the bulk solution to bind with the conjugate to alter theconfiguration of the pair of fluorescent materials and alter thefluorescence of the fluorescent materials to detect and quantify thefirst DNA.
 2. The procedure of claim 1 wherein said fluorescentmaterials are fluorescent energy transfer dyes.
 3. The procedure ofclaim 1 wherein said fluorescent materials are organic ligands oflanthanide metals.
 4. The procedure of claim 1 wherein the fluorescentmaterials are attached to the 3′ and 5′ ends of the second DNA.
 5. Aprocedure for detecting and quantifying a first DNA in bulk solutioncomprising: (a) placing in the bulk solution a conjugate comprising: asecond DNA strand complementary to at least a portion of said first DNA,said second DNA strand having a pair of fluorescent materials capable offorming a dimer attached to the ends of the second DNA strand, saidconjugate capable of moving between a more stacked configuration and amore stacked configuration to exhibit a change in fluorescence; and (b)adding the first DNA to the bulk solution to bind with the conjugate tothus alter the configuration of the pair of fluorescent materials andalter the fluorescence of the fluorescent materials in order to detectand quantify the first DNA.
 6. The process of claim 5 wherein saidfluorescent materials are fluorescent energy transfer dyes.
 7. Theprocess of claim 5 wherein said fluorescent materials are organicligands of lanthanide metals.